Study On The Molecular Mechanism Of Antidepressant Effects Of Helicid

With the accelerated living pace and pressure of modern society,the incidence of depression is rising year by year.Because the pathogenesis of depression is not clear,and depressed patients cannot fully recover with currently available drug therapy.More and more studies,therefore,are trying to find and develop drugs with good antidepressant effects from nature plants,and strive to discover biomarkers of depression and new targets for antidepressant treatment.Previous studies have found that helicid has certain experimental antidepressant effects.In this study,we used chronic unpredictable mild stress(CUMS)rat depression model to explore the antidepressant effects of helicid.Based on the whole transcriptome sequencing,we studied the differential gene expression and signaling pathways of hippocampus in CUMS rats,and explored the effects of helicid on HPA axis,cytokines,monoamine neurotransmitters and related signaling pathways,verified the function of related genes and effects of helicid in vitro.The main results are as follows:1.After rats were reared in isolation and subjected to CUMS for 6 weeks,the weight gain(p < 0.001),and sucrose preference(p < 0.001)were seen reduced compared with the control group.In the open field test(OFT),animals exposed to CUMS covered less distance(p<0.001)and exhibited a decreased number of zone crossings and rearing events(p<0.05,p<0.001)compared with the control group,and CUMS significantly prolonged the duration of immobility(p<0.01)in the forced swimming test(FST).All indicated that CUMS can significantly reduce the spontaneous activity and exploration behavior of rats,aggravate the degree of despair,and reduce weight,appetite and pleasure of rats,suggesting that the CUMS model was successfully established.When helicid(32,16,8 mg/kg)and fluoxetine(5 mg/kg)were administrated for 6 weeks,the body weight of CUMS+FLU group and Helicid group(32,16 mg/kg)was significantly increased(p<0.001),so was the sucrose preference(p<0.001,p<0.001,p<0.05).The total distance of CUMS+FLU group and Helicid group(32 mg/kg)was significantly increased(p<0.05),and the number of zone crossings in the CUMS+FLU group and the Helicid group(32 mg/kg,16 mg/kg)was significantly inceased(p<0.05,p<0.01,p<0.001).The number of rears in the CUMS+FLU group and the Helicid group(32 mg/kg)were significantly increased(p<0.05,p<0.01).Rats in CUMS+FLU group and the Helicid group(32 mg/kg)were found significantly reduced the immobility time(p<0.001,p <0.05),indicating that helicid can significantly improve the depression-like performance of CUMS model rats,suggesting helicid have a good antidepressant effect.2.Transcriptome sequencing of hippocampus revealed that there were 90 significant up-regulated and 117 down-regulated differentially expressed genes in Group C_VS_Group M.KEGG analyzed that those significant differentially expressed genes involved in 5-HT neuronal pathway,neurotrophin pathway,cell proliferation,differentiation,apoptosis and inflammatory pathways,immune function regulatory pathways,such as TNF signaling pathway,PI3K-Akt signaling pathway,Neuroactive ligand-receptor interaction,MAPK signaling pathway,JAK-STAT signaling pathway,Cytokine-cytokine receptor interaction,Antigen processing and presentation,and these accord with current understanding of the pathophysiological mechanisms of depression.Group C_VS_Group M ? Group H_VS_Group M showed 13 up-regulated and 27 down-regulated differentially expressed genes overlapped;q RT-PCR confirmed that helicid can significantly down-regulate the ARSI,PNOC,NCALD genes highly expressed in CUMS group;the signaling pathways involved are Cytokine-cytokine receptor interaction,Neuroactive ligand-receptor interaction,Antigen processing and presentation.These proved the genes and signaling pathways involved in the antidepressant effects of helicid.Fluoxetine can exert antidepressant effects by regulating the function of the immune system,such as HTLV-I infection,Antigen processing and presentation,Autoimmune thyroid disease signaling pathways and others.Helicid has a broader antidepressant mechanism than the positive drug fluoxetine.3.In vivo experiments verified the results of transcriptomics studies.We found that the serum CORT level was decreased(p<0.05,p<0.01)of Helicid group(32 mg/kg)and CUMS+FLU group(5 mg/kg);TNF-?,IL-6 and IL-1? was found decreased(p<0.05,p<0.01,p<0.05)in the rat hippocampus of the Helicid group(32 mg/kg),and the level of IL-6 in Helicid group(16 mg/kg)was decreased(p<0.05).TNF-?,IL-6 and IL-1? levels in CUMS+FLU group(5 mg/kg)were significantly decreased(p<0.01,p<0.001,p<0.01).The 5-HT levels were significantly increased(p <0.01,p<0.001)of Helicid group(32 mg / kg)and CUMS+FLU group(5 mg/kg),and the content of 5-HT1 A receptor in Helicid group(32 mg/kg)increased(p<0.05);p-ERK1/2/ERK1/2,p-CREB/CREB and BDNF were up-regulated(p<0.001,p<0.01,p<0.01)of Helicid group(32 mg/kg)and CUMS+FLU group(p<0.001,p<0.001,p<0.001).The results suggested that helicid can inhibit HPA axis,down-regulate the expression of inflammatory cytokines,increase monoamine neurotransmitters and their receptors,and promote nervous plasticity;its effect is related to neurotrophic factors,and function through the ERK-CREB-BDNF signaling pathway.These are consistent with the transcriptome results.4.In vitro experiments investigated NCALD gene function.ROS,GFAP,i NOS,COX-2(p<0.01)and cytokines TNF-?,IL-1? and IL-6(p<0.001)were highly expressed in LPS-induced C6 cell inflammation model;GDNF,p-ERK,p-AKT(p<0.01)were down-regulated,and can be significantly reversed by si RNA silenced NCALD gene,indicating that NCALD gene may participate in cellular inflammatory response through oxidative stress,neurotrophic factors,and cytokine expression.Helicid can reduce the expression of NCALD gene and the expression of ROS,GFAP,i NOS,COX-2(p<0.01)and cytokines TNF-?,IL-1? and IL-6(p<0.01),while improve the expression of GDNF,p-ERK,p-AKT(p<0.05).The overexpression of NCALD gene can significantly reverse the effect of helicid,indicating that helicid has protective effect on cellular inflammatory response,and its mechanism may be related to NCALD gene.This study found that helicid can significantly improve the depression-like performance of CUMS model rats.Its mechanism involved increasing the content of monoamine neurotransmitters and receptors,down-regulating the expression of inflammatory cytokines and acting through the ERK-CREB-BDNF signaling pathway.In vitro experiments confirmed that NCALD gene is involved in cellular inflammatory response,and helicid plays a protective role in cellular inflammatory response by regulating NCALD gene function.This study is the first to systematically study helicid that will be an antidepressant,which will provide a reliable pharmacological basis for expanding the clinical indications of helicid,and it also enriches the research foundation of natural antidepressants.